Table 1.

Troubleshooting and helpful tips.

Issue	Appearance	Solution
Short nipple (Fig. 2)	Nipple has low profile – hard to grab	It is sometimes easier to hold the skin near the nipple and target the center of the nipple with the needle. The needle will likely dive under the skin. Pulling up slowly may reveal the nipple to be slightly over the tip of the needle and give room to grab and pull it the rest of the way onto the needle. Be very careful when diving below the skin about the angle of the needle. It is easy to inadvertently get a fat pad injection by stabbing at the wrong angle.
Fat pad injection (Fig. 2)	Swollen around nipple and possibly in nipple itself – easiest to see if color is added to injection solution	If nipple is swelling with first few ul injected, remove needle, and attempt to insert again with more care taken of angle. Begin injection again and watch for further swelling. If swelling continues, abandon attempt. It is very rare to successfully inject a nipple that has started out as a fat pad injection.
Wounds/scabbing	Open wound or scabbing near injection site of EtOH solution	Rats are more likely than mice to develop wounds or scabbing near the injection area. If wounds are found, apply triple antibiotic ointment to open wounds but leave scabbed wounds alone. Applying ointment to scabs can increase likelihood animal will bother the scab and remove it. Check every 1–2 days until healed depending on severity of wound. Carprofen should be given until healed even if beyond normal window.
Inject alternating glands	N/A	Larger injection volumes in rats make it more likely to cause skin abrasions if injecting consecutive glands. For least likelihood of trauma to injection area, alternate glands injected within a single session (i.e. inject #1, 3, 4 and 6 rather than #1–4). Spacing between third (#3 and #9) and fourth (#4 and #10) gland pairs allows injection of both of these glands in one session.